GSM7712908: RPE-1_SA1_ip_1; Homo sapiens; ChIP-Seq
To fix cells, 16% methanol-free formaldehyde solution (Pierce) was added directly to the culture medium to a final concentration of 1% and incubated at room temperature for 10 min with gentle agitation. Formaldehyde was quenched with glycine (final =125 mM) for 5 min at room temperature. Cells were washed 3 times in 1x ice-cold phosphate buffered saline (PBS) containing 1mM PMSF, 1x protease inhibitory cocktail (PIC) (Roche) and harvested by using a cell lifter, spun down at 2000 rpm for 15 min (RPE-1)/1200 rpm for 8 min (CHM13) at 40C. Pellet was washed in 2 ml Buffer A2 (15 mM HEPES, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1% SDS, 0.5% N-lauroylsarcosine, 1mM DTT, 1 mM PMSF, 1x PIC, spun at 2,000/1800 rpm for 5/8 min at 40C (RPE-1 and CHM13 respectively). For RPE-1 cells, 8 x 10e6 cells were re-suspended in 1.8 mL Buffer A2 and distributed in 6 nos. 1.5 mL Bioruptor® tubes. Cells were sonicated using Bioruptor bath sonicator for 2 rounds of 7 cycles (1 cycle: 30" ON, 30" OFF) at 40C with 1 min of incubation on ice followed by vortexing between each round. 1 x 10e7 CHM13 cells, were resuspended in 1 mL of buffer A2 and aliquoted into 1.5 mL tubes (Covaris®). Cells were sonicated for 8 min (Power: 140, Duty factor: 6, 200 bursts/cycle) on Covaris S220 ultrasonicator. Sonicated chromatin was pooled and spun at 14,000 rpm for 15 min, 40C and the pellet was discarded. All steps from here on were performed in Low protein binding microcentrifuge tubes (Thermo Scientific, 90410). Bead preparation and antibody binding: 30-50 µL of DynabeadsTM Protein G (Invitrogen, 10003D) were used per ChIP. Beads for each ChIP were distributed in 1.5 mL tubes, washed twice in 1x PBS and once 0.5% bovine serum albumin (BSA)-1x PBS. For antibody binding, each tube of beads was resuspended in 800 µL of 0.5% BSA-1x PBS and incubated with 5 µg of each antibody for 3 h at 40C on a nutator at low speed. Antibody bound beads were washed once in 0.5% BSA-1x PBS. Pre-clearing: DynabeadsTM Protein G beads were prepared as before and 10 µL of blocked beads were used to pre-clear ~100 µg of chromatin for 3 h at 40C. 10% of pre-cleared chromatin was removed as Input and flash frozen in liquid nitrogen. Chromatin immunoprecipitation: ~75-100 µg of pre-cleared chromatin (amounts varied across replicates but constant for each antigen in a single experiment) was added to antibody-bead conjugates, volume was made up to 800 µL with Buffer A2 and incubated overnight at 40C on a nutator at low speed. The following day, beads were collected using DynamagTM (Invitrogen) and supernatant was discarded. Beads were washed 3 times in low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100), 3 times in high salt buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100) and once in LiCl buffer (20 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Triton X-100, 0.1% NP-40, 0.5% sodium deoxycholate). Washes were 10 min each at 40C. Chromatin elution and de-crosslinking: 100 µL elution buffer (EB) (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and 100 µL TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) were added to beads in each tube and incubated at 650C for 15 min. Supernatant was collected and the step was repeated once more. Input volume was made up to 400 µL with 1:1 EB:TE and treated similarly. NaCl (final = 210 mM) and 20 µg (2 µL) Proteinase K (Invitrogen) were added to each tube of eluted chromatin overnight at 650C on a thermomixer at 600 rpm. 10 µg of RNaseA (Invitrogen) to each tube and incubated at 370C for 1h. DNA purification: DNA was extracted with equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and the aqueous layer was transferred to new tube containing NaCl (final = 200 mM) and 2 µL GlycoBlueTM (ThermoFisher Scientific, AM9515). 750 µL of ice-cold 100% ethanol was added, and tubes were incubated at -800C for 1 h, centrifuged at 14,000 rpm for 30 min at 40C, DNA pellet was washed once with 70% ethanol and air-dried. Finally, DNA pellets were re-suspended in ultrapure distilled water (Invitrogen). Libraries were prepared using the KAPA HTP Library Prep Kit for Illumina (Roche, KK8234) and Bioo Scientific NEXTflex DNA barcodes (Perkin Elmer, NOVA-514104). The resulting libraries were purified using the Agencourt AMPure XP system (Beckman Coulter, A63882) then quantified using a Bioanalyzer HS DNA kit (Agilent Technologies, 5067-4626) and a Qubit fluorometer (Life Technologies) with Qubit dsDNA HS assay kit (Invitrogen, Q32851). Post amplification size selection (275-700 bp) was performed on all libraries using a PippinHT (Sage Science) using 1.5% cassette (HTC1510). Libraries were pooled, requantified, and sequenced as 150 bp paired reads on a mid-output flow cell using the Illumina NextSeq 500 instrument.